Digital Spatial Profiling of Individual Glomeruli From Patients With Anti-Neutrophil Cytoplasmic Autoantibody-Associated Glomerulonephritis

Abstract

We previously showed that the rupture of Bowman’s capsule (BC) promotes the progression of crescentic glomerulonephritis by enhancing the entry of CD8+ T cells into the glomeruli. In the present study, we utilized digital spatial profiling to simultaneously profile the altered abundances of the messenger RNA (mRNA) transcripts and proteins in the glomerular and periglomerular areas of four biopsy samples of anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis (ANCA-GN) and two biopsy specimens of minimal change disease (MCD) controls. The paraffin-embedded biopsy samples were stained with collagen IV, CD45, and SYTO 13 to distinguish the glomeruli with periglomerular infiltration but intact BC, with focal BC rupture, and with extensive rupture of BC and glomeruli without crescent formation and leukocytic infiltration in ANCA-GN. By assessing multiple discrete glomerular areas, we found that the transcript expression levels of the secreted phosphoprotein-1 and its receptor CD44 were upregulated significantly in the glomeruli with more severe ruptures of BC, and their expression levels correlated positively with the fibrotic markers. We also found that both alternative and classic complement pathways were activated in the glomeruli from patients with ANCA-GN. Furthermore, M1 macrophages were involved mostly in the early stage of BC rupture, while M2 macrophages were involved in the late stage and may contribute to the fibrosis process of the crescents. Finally, loss of glomerular cells in ANCA-GN was likely mediated by apoptosis. Our results show that digital spatial profiling allows the comparative analysis of the mRNA and protein profiles in individual glomeruli affected differently by the disease process and the identification of potential novel mechanisms in ANCA-GN.

Assessment of spatial transcriptomics for oncology discovery

“… 98 Another technology, digital spatial profiling (DSP), allows for the localization of transcripts and proteins within small pre-selected regions of interest, making it better suited for focused hypothesis testing than de novo discovery. 99 Selecting the best technology for a given question is an important consideration when designing these experiments.…”

Analysis and Validation of Antineutrophil cytoplasmic antibody-associated vasculitis with renal injury of the ferroptosis-related gene CD44 and Pan-Cancer

“…The more seriously injured glomeruli with BC rupture, however, were shown to have a signi cant in ow of CD8 + T cells, along with podocyte degeneration and the onset of a catastrophic, fast progressing glomerulonephritis 34 . The mRNA expression levels of secreted phosphoprotein-1 and its receptor CD44 have been discovered to be considerably elevated in glomeruli with more extensively ruptured BC in AAV, and their expression levels are strongly linked with brosis indicators 35 . According to these ndings, CD44 may be linked to CD8 + T cell in ltration in AAV with renal injury.…”

Monoclonal Gammopathy of Renal Significance: A Molecular Middle Earth between Oncology, Nephrology, and Pathology

“…The application of digital spatial profiling is giving promising results in the study of hematological disorders due to its unprecedented capability of discovering subtle transcriptomic modifications in small regions of tissue that approximate the single-cell analysis [ 74 ]. This already found application on renal biopsies in different settings, such as ANCA-associated GN [ 75 ] and graft rejection [ 76 ], even showing interesting molecular modifications in murine models of acute kidney damage, elucidating region-specific loss of differentiation markers and their re-expression during repair [ 77 ]. This has also been applied to investigate the complex interaction between inflammatory cells and surrounding renal structures, such as in the case of LYVE1+ macrophages in the medulla human kidney and its putative protective role against fibrosis or the immunomodulatory role of macrophage Trem2 in damaged tissue [ 78 , 79 ].…”